**3. New technologies in genetic engineering**

The traditional methods of genetic manipulation are time-consuming when dealing with industrial strains, as they usually have multiple copies for each gene. Gene editing by using the CRISPR-Cas9 technology is faster to cause multiple gene deletions and introducing punctual changes. New tools as genome editing and genome synthesis are building up a new era for the synthetic biology. Their application for yeasts of biotechnological interest will change the paradigm in the ways we approach the use of those microorganisms for a particular task, as their abilities can be tailored from the beginning to the end.

#### **3.1 Wine yeasts genome editing by CRISPR-Cas9**

Industrial yeast strains are usually diploid or polyploidy with a more complex genetic background than the well-studied haploid laboratory strains. Using a traditional PCR-based technique for the genetic manipulation of industrial strains is normally very time consuming, laborious and often even impossible [55]. In recent years, the development of an alternative genome editing approach, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR–Cas9) system, can help to solve the problem [55, 56].

At first, CRISPR-Cas system was discovered to provide an immunological weapon for bacteria and archaea against the attack by viruses (bacteriophages) or

#### *Genetically Modified Yeasts in Wine Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.98639*

invading mobile genetic elements [57, 58]. The CRISPR system from *Streptococcus pyogenes* has been well characterized and it is still the most widely used in yeast genetic engineering. Two elements are necessary for the correct operation of the CRISPR-Cas system. The Cas9, a 160 kilodalton protein, is a RNA-mediated endonuclease that recognizes a 3-nucleotide protospacer adjacent motif (PAM), NGG (where N is any nucleotide, followed by two guanines (G)), and makes doublestranded breaks (DSBs) between the third and fourth nucleotides upstream to the PAM site. Another key component is a single guide RNA (sgRNA) that guides Cas9 to target sites. The sgRNA derives from a duplex of two RNA molecules: a CRISPR targeting RNA (crRNA), which is complementary to the target, and a trans-activating CRISPR RNA (tracrRNA). The first 20 base pairs at 5′ end of crRNA binds to the complementary genomic target, and PAM site must be found immediately at 3′ end of the desired locus in genome [59]. The sgRNA has a concrete secondary structure to recruit Cas9 to establish a functional complex. Following the guide of sgRNA, Cas9 target the genome specific sequence with PAM and cut double-strand DNA [60]. DSB must be repaired by cells via nonhomologous end-joining (NHEJ) or homologous recombination (HR). Normally, NHEJ repair is considered to generate small nucleotide insertions or deletions, and HR is used for precise modifications with the existence of donor DNA (**Figure 1**).

CRISPR-Cas9 genome-editing technology was first applied in *S. cerevisiae* in 2013 [56]. Vigentini and co-authors successfully established the CRISPR-Cas9 system in commercial wine strains EC1118 and AWRI1796. In this study, *CAN1* gene encoding for an arginine permease was deleted, in order to generate strains with reduced urea production [61] (see above about EC). The resulting *can1*Δ mutants were characterized by decreased urea production (18 and 35.5% compared to EC1118 and AWRI1796, respectively) under micro-winemaking conditions, in Chardonnay and Cabernet Sauvignon grape musts. Recently, Wu et al. [62] use CRISPR-Cas9 system for over-expressing the *DUR3* gene in a previously engineered rice wine strain with *CAR1* gene disrupted and *DUR1,2* genes over-expressed [63]. *CAR1* encodes an arginase responsible for the arginine cleavage generating urea. Urea can be hydrolysed into NH3 and CO2 by urea amidolyase (encoded by *DUR1,2*), and *DUR3* encodes a transporter that transfers urea from the fermentation broth to yeast cells when nitrogen source is insufficient. A laboratory fermentation experiment of Chinese rice wine shows that the CRISPR-Cas9 engineered strain reduces urea and EC concentrations by 92% and 85%, respectively, compared with those of the original strain (N85).

In another work, a polygenic analysis combined with CRISPR-Cas9-mediated allele exchange reveals novel *S. cerevisiae* genes involved in the production of 2-phenylethyl acetate (PEA). PEA is a desirable flavor compound that provides alcoholic beverages a rose and honey aromas. With the mentioned approach, unique alleles of the *FAS2* gene (encodes de α subunit of fatty acid synthase) and a mutant allele of *TOR1* (growth regulator in response to nitrogen sources) were identified to be responsible for high PEA production. Then, using CRISPR-Cas9, wild type alleles were replaced with mutant ones in commercial wine strains. PEA production in these yeasts increased by 70% [64].

In a recent study, Walker and co-authors [65] used CRISPR-Cas9 system to introduce selected mutations in *SUL1* and *SUL2* genes in wine strains EC1118. These genes encoded two high-affinity sulfate transporters. Under nitrogen limitation, sulfate contributes to hydrogen sulfide (H2S) production, a common wine fault with a rotten-egg odor. The introduced mutations affect protein-structure function of Sul1 and Sul2 and shown to reduce H2S accumulation during fermentation in Riesling juice or a chemical defined grape juice.

#### **Figure 1.**

*Overview of the CRISPR-Cas9 system. The Cas9 interacts with sgRNA and form a complex. The Cas9-sgRNA complex binds to the target DNA sequence upstream of PAM site. The Cas9 protein cleaves DNA sequence complementary to the 20 bp guide sequence producing a double-strand break (DSB). After the nuclease cuts the DNA can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR).*

In *S. cerevisiae*, glycerol is a key polyol that reduces osmotic stress and controls intracellular redox balance. Muysson et al. [66] established a CRISPR-Cas9-based genome-editing approach to investigate the Stl1p (a H+ /glycerol symporter) role in ice wine fermentations. In this study, *STL1* gene was deleted in *S. cerevisiae* K1-V1116 strain. During ice wine fermentation, the *stl1*Δ mutant presents increased glycerol and acetic acid production compared to the original strain, suggesting that Stl1 plays an important role in these conditions. In a study carried out by van Wyk et al. [67], CRISPR-Cas9 system was used to increase glycerol and ester

#### *Genetically Modified Yeasts in Wine Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.98639*

production in the AWRI1631 wine yeast strain. First, two newly strains were created, one that overexpressed *GPD1* and the other that overexpressed *ATF2*. *GPD1* encodes a glycerol-phosphate dehydrogenase involved in glycerol formation; and *ATF2* encodes an alcohol acetyltransferase which promotes condensation between alcohols and acetyl-CoA resulting in more acetate esters produced, important for flavor in fermented beverages. Mating these engineered strains, the authors obtained a new strain that overexpressed *GPD1* and *ATF1* genes. Riesling wine from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain.

Vallejo and co-authors [68] described recently that nutrient signaling pathway genetic manipulation can be a good target of yeast performance improvement during winemaking. Using CRISPR-Cas9 system in commercial wine strain EC1118, *PDE2* gene encoding for a phosphodiesterase was deleted. Pde2 is a cAMP degrading enzyme whose deletion increases cAMP-dependent protein kinase A (PKA) activity. The resulting *pde2*Δ mutant showed increased fermentation speed compared to EC1118, in red grape juice. The results suggest that Pde2p inactivation is a way to increase fermentative performance.

#### **3.2 Synthetic genome engineering**

Synthetic biology seeks to standardize and modularize the design and engineering of organisms to achieve novel functions, or to construct genomes or even organisms from the ground up using rational laboratory procedures or automation [69]. Synthetic biology is regarded as the most exciting interdisciplinary science of the twenty-first century, with applications in yeast biotechnology and strain development, among other things. Given yeast's importance in the fermentation industry as well as its role as an experimental research model organism in the advancement of Synthetic Biology, the wine industry will be impacted by the outcomes of this field. Synthetic Biology techniques are already being applied to the production of better wine yeast strains [70, 71].

In 1996, the 14 Mb genome of a haploid laboratory strain (S288c) of *S. cerevisiae* was sequenced for the first time, revealing that its 16 chromosomes encode 6000 genes, of which 5000 are non-essential. In 2009, the first synthetic yeast genome project (Sc. 2.0 project) was launched to redesign and chemically synthesize a slightly modified version of the *S. cerevisiae* S288c strain genome. This project allows to find answers to a broad range of questions about fundamental properties of chromosomes, genome organization, gene content, RNA splicing mechanism, the role of small RNAs in yeast biology, the distinction between prokaryotes and eukaryotes, and genome structure and evolution [72]. The Sc2.0 genome was designed to contain specific base substitutions inside some of the ORFs to accommodate desirable enzyme recognition sites or deletions of undesirable enzyme recognition sites. All TAG stop codons were recoded to TAA to free up one codon for future inclusion of unusual amino acids; all repetitive and dispensable sequences were omitted; and all tRNA genes were relocated to a novel neochromosome.

In 2011, the first step toward building the ultimate yeast genome was taken with the construction of synthetic chromosome arms [73]. In 2014, *S. cerevisiae* became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome, the chromosome 3 [74]. In 2017, six redesigned yeast chromosomes were completed [72]. In 2018, 16 natural chromosomes of *S. cerevisiae* were successfully fused into a single chromosome, like in prokaryotic cells, and the artificial *S. cerevisiae* still has normal cellular functions [75]. These works blur the lines between natural and artificial life, pointing to a near-future for custom-designed yeast to fulfill all the customers' needs.

#### *Grapes and Wine*

Wine yeast strain development is well positioned to benefit from technological advances made with the genetic and genome engineering of non-wine strains of *S. cerevisiae*. For example, the first "synthetically engineered" wine yeast reveals a whiff of raspberries in an experimental Chardonnay wine. *S. cerevisiae* AWRI1631 wine strain was equipped with a biosynthetic pathway, including four separate enzymatic activities, to produce the highly desirable raspberry ketone (4-(4-hydroxyphenyl)butan-2-one) [76].
